Research article
5703
An essential role for Fgfs in endodermal pouch formation
influences later craniofacial skeletal patterning
Justin Gage Crump1,*, Lisa Maves1, Nathan D. Lawson2, Brant M. Weinstein3 and Charles B. Kimmel1
1
Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254, USA
Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA
3
Laboratory of Molecular Genetics, NICHD/NIH, Bethesda, MD 20892, USA
2
*Author for correspondence (e-mail: [email protected])
Accepted 18 August 2004
Development 131, 5703-5716
Published by The Company of Biologists 2004
doi:10.1242/dev.01444
Summary
Fibroblast growth factor (Fgf) proteins are important
regulators of pharyngeal arch development. Analyses of
Fgf8 function in chick and mouse and Fgf3 function in
zebrafish have demonstrated a role for Fgfs in the
differentiation and survival of postmigratory neural crest
cells (NCC) that give rise to the pharyngeal skeleton. Here
we describe, in zebrafish, an earlier, essential function for
Fgf8 and Fgf3 in regulating the segmentation of the
pharyngeal endoderm into pouches. Using time-lapse
microscopy, we show that pharyngeal pouches form by the
directed lateral migration of discrete clusters of
endodermal cells. In animals doubly reduced for Fgf8 and
Fgf3, the migration of pharyngeal endodermal cells is
disorganized and pouches fail to form. Transplantation and
pharmacological experiments show that Fgf8 and Fgf3 are
required in the neural keel and cranial mesoderm during
Introduction
The cartilages and bones that form the skeleton of the face and,
in mammals, the middle ear, are derived from a specialized
population of ectomesenchyme, the cranial neural crest (Le
Douarin, 1982; Weston et al., 2004). Cranial neural crest cells
(NCC) originate adjacent to neural ectoderm and migrate in
three streams (mandibular, hyoid and branchial) to form seven
pharyngeal arches. Segmentation of NCC into distinct streams
is coupled to the segmentation of the hindbrain into
rhombomeres (R1-7) (Kontges and Lumsden, 1996). NCC that
contribute to the formation of the mandibular arch delaminate
adjacent to posterior midbrain-R2 and do not express Hox genes,
whereas NCC of the hyoid and branchial arches originate next
to R4 and R6-R7, respectively, and are Hox-positive (Schilling
and Kimmel, 1994; Trainor and Krumlauf, 2001).
Fibroblast growth factors (Fgfs) are a family of extracellular
signaling molecules that have been implicated in diverse facets
of vertebrate craniofacial development. Fgf8 from the oral
surface ectoderm induces patterns of gene expression in adjacent
mandibular mesenchyme, subdividing the mandibular arch into
rostral odontogenic and caudal skeletogenic fields (Tucker et al.,
1999) and controlling the position of the jaw joint (Wilson and
Tucker, 2004). In addition to functions in mandibular arch
development, Fgfs have roles in the development of cartilages
early somite stages to promote first pouch formation. In
addition, we show that animals doubly reduced for Fgf8
and Fgf3 have severe reductions in hyoid cartilages and the
more posterior branchial cartilages. By examining early
pouch and later cartilage phenotypes in individual animals
hypomorphic for Fgf function, we find that alterations in
pouch structure correlate with later cartilage defects. We
present a model in which Fgf signaling in the mesoderm
and segmented hindbrain organizes the segmentation of the
pharyngeal endoderm into pouches. Moreover, we argue
that the Fgf-dependent morphogenesis of the pharyngeal
endoderm into pouches is critical for the later patterning
of pharyngeal cartilages.
Key words: Pouch, Pharyngeal endoderm, Cartilage, Neural crest,
Segmentation, Fgf8, Fgf3, acerebellar, GFP, Zebrafish
derived from the hyoid and branchial arches and in the formation
of pharyngeal pouches. Pouches are outpocketings of the
pharyngeal endoderm that interdigitate with the crest-derived
pharyngeal arches. Fgf8neo/– mice, which are hypomorphic for
Fgf8, display a range of craniofacial abnormalities that include
reductions in cartilages and bones derived from all pharyngeal
arches and disorganized endodermal pouches (Abu-Issa et al.,
2002). Likewise, in the zebrafish acerebellar (ace) mutant, a
strong loss-of-function mutation of fgf8 (hereafter referred to as
fgf8–), hyoid cartilage is reduced and pouches are misshaped
(Draper et al., 2001; Reifers et al., 1998; Roehl and NussleinVolhard, 2001).
Increasing evidence suggests that signals from the pharyngeal
endoderm pattern the bones and cartilages of the pharyngeal
arches. Analysis of casanova (cas) mutant zebrafish, which
make no endoderm (Alexander et al., 1999), suggest that
endoderm is required for the development of all pharyngeal
cartilages (David et al., 2002). In tbx1 (van gogh) mutant
zebrafish, pharyngeal pouches are largely absent and cartilages
are misshaped and fused with those of adjacent arches
(Piotrowski and Nusslein-Volhard, 2000), suggesting that
pouches contribute to the segmentation of NCC into distinct
arches. In addition, transplantation experiments in chick show
that foregut endoderm is both necessary and sufficient to induce
the shape and orientation of pharyngeal skeletal elements (Couly
5704 Development 131 (22)
et al., 2002). One role of pharyngeal endoderm may be to locally
promote the survival of skeletogenic NCC (Crump et al., 2004).
Fgf3 has been shown to be required in pouch endoderm for the
survival of skeletogenic NCC of the hyoid and branchial arches
(David et al., 2002; Nissen et al., 2003), and a similar NCC
survival-promoting role for endodermal Fgf8 has been proposed
but not proven in zebrafish (Walshe and Mason, 2003a). Clearly,
understanding how pharyngeal endoderm develops is critical for
understanding the later development of the pharyngeal skeleton.
In this study, we investigate an earlier role for Fgfs in the
formation of pharyngeal pouches. Whereas in fgf8– animals
pharyngeal pouches are variably misshaped, we find that
reducing both Fgf8 and Fgf3, by injecting fgf8– animals with an
fgf3 morpholino (fgf3-MO), leads to a complete failure of pouch
formation. We use time-lapse microscopy to show that
pharyngeal pouches form by the directed lateral migration of
periodic clusters of endodermal cells. In fgf8–; fgf3-MO animals,
pharyngeal endodermal cells are present but their lateral
migration is disorganized and discrete pouches fail to form. We
use the Fgf receptor-inhibiting drug SU5402 to show that Fgf
signaling is required during early somite stages for first pouch
formation. At these stages, fgf8 is expressed in the head in lateral
mesoderm (Reifers et al., 2000) and in midbrain-hindbrain
boundary (MHB)-R2 and R4 domains of the hindbrain (Maves
et al., 2002; Reifers et al., 1998). Starting at 4-somites (11.3
hours post-fertilization), fgf3 expression overlaps fgf8 expression
in neural MHB and R4 domains (Maves et al., 2002; Walshe et
al., 2002), and mesodermal and neural Fgf expression domains
are in close proximity to developing pharyngeal endoderm. At
later stages, fgf3 and, to a lesser extent, fgf8 are expressed in the
pharyngeal pouches (David et al., 2002; Walshe and Mason,
2003a). However, we use mosaic analysis to show that Fgfs are
required in mesodermal and neural domains, and not in the
pharyngeal endoderm, to rescue pharyngeal arch structure in
fgf8–; fgf3-MO animals. Thus, we find an essential, Fgfdependent function of the brain and head mesoderm in
controlling segmentation of the pharyngeal endoderm into
pouches.
In addition to their requirement in pouch formation, we find
that Fgf8 and Fgf3 have redundant, essential functions in
pharyngeal cartilage development. In fgf8–; fgf3-MO animals,
hyoid and branchial cartilages are largely absent and mandibular
cartilages are reduced. By imaging pouch structure early and
cartilage structure later in individual sides of animals in which
Fgf signaling has been manipulated, we find that altered pouches
correlate with later rearrangements of the cartilage pattern. This
analysis suggests that pharyngeal pouch structure is a critical
determinant of the pharyngeal cartilage pattern. We present a
model in which an earlier function of Fgfs in pouch formation,
in addition to their well-documented role as pouch-secreted
survival factors later in development, contributes to the diversity
of craniofacial phenotypes seen in fgf8 mutants.
Materials and methods
Zebrafish lines and morpholinos
Zebrafish (Danio rerio) were raised and staged as previously described
(Kimmel et al., 1995; Westerfield, 1995). Time (hpf) refers to hours
post-fertilization at 28.5°C. The wild-type line used was AB.
Homozygous acerebellarti282a (ace) mutant embryos were scored by
their loss of the cerebellum or loss of midbrain pax2a expression (Brand
Research article
et al., 1996; Reifers et al., 1998). fli1-GFP albino transgenic fish are the
same as TG(fli1:EGFP)y1; albb4 (Lawson and Weinstein, 2002) and
H2A.F/Z:GFP transgenic fish are as described (Pauls et al., 2001).
The fgf8 MOs E2I2 and E3I3 (Draper et al., 2001) were used at 0.5
mg/ml each. To generate fgf8–; fgf3-MO embryos, we pressure-injected
fgf8– embryos at the one-cell stage with 5 nl of the fgf3B (1.0 mg/ml)
+ fgf3C (0.25 mg/ml) MO combination. As previously described
(Maves et al., 2002), this fgf3 MO dose gave highly reproducible fgf8–;
fgf3-MO phenotypes, scored as either the lack of ears or the lack of R5
krox20 staining.
Phenotypic analysis
Alcian Green staining was performed as described (Miller et al., 2003).
For flat-mount dissections, Alcian-stained animals were digested for 1
hour in 8% trypsin at 37°C and transferred to 100% glycerol. Cartilages
were dissected free from surrounding tissues with fine stainless-steel
insect pins and photographed using a Zeiss Axiophot 2 microscope.
Image background was cleaned up with Adobe Photoshop. For
immunocytochemistry, embryos were prepared as described (Maves et
al., 2002). Antibodies were used at the following dilutions: rabbit antiGFP, 1:1000 (Molecular Probes); Zn8, 1:400 (Fashena and Westerfield,
1999; Trevarrow et al., 1990), goat anti-rabbit Alexa Fluor 488 and antimouse Alexa Fluor 568, both 1:300 (Molecular Probes).
The following cDNA probes were used: dlx2 (Akimenko et al.,
1994); krox20 (Oxtoby and Jowett, 1993); pax2a (Krauss et al., 1991);
axial (Odenthal and Nusslein-Volhard, 1998); nkx2.7 (Lee et al., 1996);
pea3 (Brown et al., 1998). Probe syntheses and whole-mount in-situ
hybridizations were performed as previously described (Hauptmann
and Gerster, 1994; Jowett and Lettice, 1994).
SU5402 treatment
fli1-GFP embryos were manually dechorionated and incubated in 40 µl
of EM with 0.4 mM SU5402 (Calbiochem) in agar wells. SU5402 was
diluted from a 40 mM stock in DMSO. After 1- or 4-hour incubations,
embryos were washed vigorously in EM. For 4-hour incubation
experiments, sibling controls were fixed and processed for in-situ
hybridizations with pea3.
Transplantations
Transplant techniques were as described (Maves et al., 2002). For
endoderm transplants, donor embryos were injected at the 1-cell stage
with an ‘Alexa568’ mixture of 2% Alexa Fluor 568 dextran and 3%
lysine-fixable biotin dextran (10,000 Mr, Molecular Probes) along with
activated Taram-A receptor (TAR*) RNA prepared according to David
et al. (David et al., 2002). At 40% epiboly (ca. 4 hpf) donor TAR* tissue
was moved to the margins of fgf8–; fgf3-MO; fli1-GFP host embryos.
For neural and mesodermal transplants, donor embryos were injected
at the 1-cell stage with Alexa568. For neural transplants, donor tissue
was taken from the animal cap at shield stage (ca. 6 hpf) and moved to
a position approximately two germ ring widths from the margin and
70° from dorsal in fgf8–; fgf3-MO; fli1-GFP hosts (Maves et al., 2002).
For mesodermal transplants, donor tissue was taken from the margin at
50% epiboly (ca. 5 hpf) and moved to the margins of fgf8–; fgf3-MO;
fli1-GFP hosts (Kimmel et al., 1990). All hosts were screened using a
fluorescence stereomicroscope at 34 hpf, and only hosts with
substantial, tissue-specific contributions to the pharyngeal endoderm,
hindbrain or cranial mesoderm were used for subsequent analysis. In
addition, the mesodermal transplant technique produced six embryos
with contributions to both the hindbrain and cranial mesoderm. In order
to control for variability in the effectiveness of the fgf3-MO, only fgf8–;
fgf3-MO; fli1-GFP hosts in which the ear was missing in at least one
side were used for the analysis. In control transplants, mutant siblings
in which donor tissue did not contribute to head tissues, we never
observed the presence of an ear on only one side. At 34 hpf, confocal
images of select host embryos were analyzed for pharyngeal arch
structure. Rescue of arch structure was scored as complete if the first
pouch and mandibular and hyoid arches were indistinguishable from
Fgfs pattern pouches and cartilages 5705
wild type, and rescue was scored as partial for all other embryos with
arch structure subjectively more organized than in fgf8–; fgf3-MO; fli1GFP controls.
Time-lapse analysis and confocal imaging
For the endoderm movies (see Movies 3-6 in supplementary material),
pharyngeal endoderm was labeled by transplanting donor tissue injected
with a mixture of Alexa568 and TAR* into GFP hosts. Embryos were
selected for large fractions of labeled donor endoderm and bright GFP
fluorescence using a Leica MZ FLIII fluorescence stereomicroscope.
After manual dechorionation and anesthetization with buffered ethylm-aminobenzoate methane sulfanate (MESAB) (Westerfield, 1995),
embryos were transferred to 0.2% agarose in embryo media (EM) with
10 mM HEPES and MESAB and then mounted onto a drop of 3%
methylcellulose on a rectangular coverslip with three superglued #1
square coverslips on each side. A ring of vacuum grease was added
around the embryo to make an airtight seal upon addition of the top
coverslip. A heated stage kept the embryos at 28.5°C. Approximately
80 µm Z-stacks at 2 µm intervals were captured every 6 minutes using
a Zeiss LSM5 Pascal confocal fluorescence microscope. At each time
point, Z-stacks were projected with maximum intensity onto a single
plane. Time-lapse recordings were further processed with Adobe
Premiere. For single time point confocal sections, embryos were
mounted without vacuum grease.
Results
Fgf8 and Fgf3 are essential for the formation of
pharyngeal pouches and most pharyngeal cartilages
As previously reported (Roehl and Nusslein-Volhard, 2001),
fgf8– zebrafish had incompletely penetrant and expressive
defects in the formation of pharyngeal pouches and cartilages
(Fig. 1B,B′,F and especially Fig. 2I,J). Since the fgf8–
phenotype is relatively mild, we wondered if other Fgfs were
partially redundant with Fgf8 in patterning pharyngeal arches.
Fgf3 was a good candidate, as it has been shown to act
redundantly with Fgf8 to pattern the posterior hindbrain,
forebrain and ear (Maroon et al., 2002; Maves et al., 2002;
Phillips et al., 2001; Walshe et al., 2002; Walshe and Mason,
2003b). In addition, Fgf3 has been shown to play a role in
zebrafish pharyngeal arch development (David et al., 2002;
Nissen et al., 2003). Although fgf3-MO animals had largely
normal pouches (Fig. 1C,C′) (David et al., 2002), we found that
in fgf8–; fgf3-MO animals no pouches were made (Fig. 1D,D′).
In addition, we found that Fgf8 and Fgf3 acted redundantly
to promote pharyngeal cartilage development. In fgf3-MO
animals, the ceratobranchial (CB) cartilages, which derive
from the branchial arches located posterior to the hyoid arch,
were largely absent (David et al., 2002; Nissen et al., 2003).
However, whereas hyoid cartilages of fgf3-MO and fgf8–
animals were relatively mildly affected at 4 days (Fig. 1F,G),
nearly all of the hyoid and CB cartilages in fgf8–; fgf3-MO
animals were absent (Fig. 1H). Although reduced in size,
mandibular cartilages were patterned correctly in fgf8–; fgf3MO animals (inset to Fig. 1H).
Since fgf8–; fgf3-MO animals have both pouch and cartilage
defects, we wondered whether defects in pouch development
could be responsible for later cartilage losses. In order to study
correlations between pouch and cartilage defects in individual
animals, we took advantage of the fact that partially reducing
Fig. 1. Fgf8 and Fgf3 have redundant functions in the formation of pharyngeal pouches and cartilages. (A-D) Confocal micrographs are merged,
lateral views of cranial NCC (green: anti-GFP antibody) and endodermal pouches (red: Zn8 antibody) at 34 hpf; A′-D′ show just the red channel.
(A,A′) In wild-type fli1-GFP animals, the NCC-containing pharyngeal arches are numbered 1-7 (A) and the pouches are numbered p1-p5 (A′). A
few hours later, the sixth pouch will form and arches 6 and 7 will separate to form the final arrangement of seven arches. (B,B′) fgf8–; fli1-GFP
animals have variable defects in pouch structure (arrowhead in B′ denotes a misshapen first pouch). Whereas fgf3-MO; fli1-GFP animals have
normal pouches (C,C′), fgf8–; fgf3-MO; fli1-GFP animals lack all pouches (D,D′), although pharyngeal endoderm is still present (white line in
D′). The Zn8 antibody also recognizes cranial sensory ganglia (dots in A-C, A′-C′). (E-H) Ventral whole-mount views show Alcian-stained
pharyngeal cartilages at 4 days. As shown for wild type (E), M and PQ cartilages are derived from the mandibular, or first, arch; CH and HS are
hyoid, or second, arch cartilages, and CB1-5 cartilages are formed from the five most posterior branchial arches. fgf8– animals have relatively
mild defects in pharyngeal cartilages (F), and in fgf3-MO animals CB cartilages are lost and hyoid cartilages are misshapen (G). However, in
fgf8–; fgf3-MO animals, nearly all CB and hyoid cartilages are absent and mandibular cartilages are reduced in size (H). The inset to H is a flatmount preparation of fgf8–; fgf3-MO cartilages showing that, although reduced in size, M and PQ cartilages retain their distinctive shapes. In
E-H, asterisks denote the position of the midline neurocranium that is still present in fgf8–; fgf3-MO animals. Anterior is to the left in all panels.
M, Meckel’s; PQ, palatoquadrate; CH, ceratohyal; HS, hyosymplectic; CB, ceratobranchial. Scale bars: 50 µm in A-D; 100 µm in E-H.
5706 Development 131 (22)
Research article
Fig. 2. Correlated first pouch and hyoid cartilage
defects in animals reduced for Fgf8. Confocal
projections of fli1-GFP-labeled pharyngeal arches in
living wild-type (A) and fgf8-MO (C,E,G) animals
at 28 hpf. By this stage of wild-type development,
the mandibular (1), hyoid (2), and three branchial
arches (3, 4, 5-7) have formed. Pouches are labeled
p1-p4 (A), and white arrowheads mark the positions
of the first pouch. In fgf8-MO; fli1-GFP animals,
variable phenotypes include shape changes in the
first pouch (C,E), ectopic pouches (arrow in G), and
reductions of more posterior pouches (C,E,G).
(I) Confocal micrograph of a fixed fgf8–; fli1-GFP
animal with a similar ectopic pouch phenotype
(arrow) to the fgf8-MO; fli1-GFP animal in G; Zn8
staining (red) confirms that the non-fli1-GFPexpressing region is probably an ectopic
endodermal pouch. (B,D,F,H,J) Flat-mount
preparations of Alcian-stained mandibular and
hyoid cartilages at 4 days. As labeled in the wildtype example (B), M and PQ are mandibular (1) and
CH and HS are hyoid (2) cartilages. (C and D, E and
F, G and H) Paired images of individual animals
imaged live for fli1-GFP early and subsequently
stained for cartilage. Variable hyoid cartilage defects
(D,F,H) are correlated with earlier first pouch
defects (C,E,G) in individual fgf8-MO; fli1-GFP
animals. In H, the black arrowhead marks an
apparent ectopic hyoid cartilage that correlates with
the ectopic pouch in G. (J) Similar ectopic cartilages (black arrowhead) were seen in some fgf8–; fli1-GFP animals. Anterior is to the left and
dorsal is up. M, Meckel’s; PQ, palatoquadrate; CH, ceratohyal; HS, hyosymplectic. Scale bar: 50 µm.
Fgf function with an fgf8 morpholino (fgf8-MO), or genetically
with the fgf8 mutation, causes variably penetrant and
expressive phenotypes. We imaged pharyngeal arch structure
in live fgf8-MO; fli1-GFP and fgf8–; fli1-GFP embryos at 28
hpf and subsequently raised individuals to 4 days to examine
cartilage. The fli1-GFP transgene (Lawson and Weinstein,
2002) labels cranial NCC shortly after ventrolateral migration
and perdures as cells differentiate into the pharyngeal cartilage
elements. fli1-GFP also marks the developing vasculature but
is not expressed in the pharyngeal mesoderm or endoderm
(except for early, transient expression in the second pouch; see
below). At pharyngula stages, the endodermal pouches are
evident as non-fli1-GFP-expressing regions separating the fli1GFP-expressing NCC of the pharyngeal arches (black in Fig.
2A). In individual sides of fgf8-MO; fli1-GFP and fgf8–; fli1GFP animals we found a correlation between early arch
structure and alterations of the hyoid cartilage pattern (Fig. 2CJ and Table 1). In all sides with abnormal first pouch
morphology we observed hyoid cartilage alterations later. In
some cases, the first pouch was ‘deformed’ (Fig. 2C), invading
hyoid NCC territory, and this arch phenotype was most often
linked to a complete loss of the dorsal hyomandibular cartilage
element (Fig. 2D). In other cases, the first pouch was ‘shifted’
to a more posterior position (Fig. 2E), and this ‘shift’ was
correlated with changes in the shape and position of dorsal
hyoid cartilage (Fig. 2F). In the most striking example of
pouch–cartilage shape correlations, a small ectopic pouch, in
addition to the normal first pouch, formed in the middle of the
hyoid NCC territory (Fig. 2G), and in these same sides an
ectopic cartilage element developed later in the hyoid arch
(Fig. 2H). To confirm that the non-GFP-expressing regions
observed in live animals probably corresponded to misshapen
and ectopic pouches, and not another non-GFP-expressing
tissue such as mesoderm, we fixed and stained fgf8– animals
displaying similar GFP phenotypes with the endoderm-labeling
Zn8 antibody (Trevarrow et al., 1990). In numerous examples,
non-GFP expressing regions in fgf8– animals, of similar shapes
and positions to those observed in the live analysis, were found
to be Zn8-positive (red in Fig. 1B and Fig. 2I; and data not
shown). Lastly, whereas the majority of fgf8– animals with
normal arch morphology early had no defects in the hyoid
cartilage pattern later, we observed graded reductions of the
hyomandibular cartilage element in some sides with no pouch
defects (Table 1). Thus, Fgf8 is likely to have other functions
in cartilage development, in addition to its role in controlling
pouch development. Nonetheless, we conclude that, in contrast
to simple cartilage losses, the alterations in hyoid cartilage
shape and position observed in a subset of animals reduced for
Fgf8 are most tightly correlated with early changes in pouch
morphology.
Posterior pharyngeal pouch defects underlie
reduced arch segmentation in fgf8-MO animals
After NCC migration the third, most posterior, NCC mass
segments into the five branchial, or gill-bearing, arches from
which the five CB cartilages subsequently develop (Fig. 3A).
It has been previously shown that the segmentation of NCC
into distinct arches requires the segmentation of the pharyngeal
Fgfs pattern pouches and cartilages 5707
Table 1. Correlated pouch and cartilage phenotypes in fgf8–; fli1-GFP animals
Hyoid cartilage phenotype
Arch structure
%(n)
Wild type
HM reduced
HM loss
Wild type
No p1
Deformed p1
Shifted p1
Ectopic p1
Arch shape
74
2
6
2
5
12
59
0
0
0
0
7
33
100
0
0
0
13
2
0
88
0
0
13
Ventral loss
0
0
0
0
0
20
Shape change
Ectopic
6
0
12
100
50
40
0
0
0
0
50
7
Percentage of sides with each phenotype is listed for fgf8–; fli1-GFP animals (n=128). In 100 wild-type animals, none of these phenotypes were seen. At 33
hpf, individual sides of fgf8–; fli1-GFP mutant animals were scored under a fluorescence dissecting microscope for pharyngeal arch structure. Hyoid arches of
mutant sides were scored as percentage of the total having the following morphology: wild type, missing the first pouch (no p1), deformed first pouch (deformed
p1), shifted first pouch (shifted p1), ectopic first pouch in addition to normal first pouch (ectopic p1), and miscellaneous defects that affected arch shape but not
first pouch structure (arch shape). Individuals were subsequently grown to 4 days to examine hyoid cartilage structure. Percentages of each arch structure
category having the following hyoid cartilage phenotypes are shown: wild type, reduction of HM (HM reduced), loss of HM (HM loss), loss of ventral cartilage
(ventral loss), rearrangement of the cartilage pattern (shape change), and ectopic cartilage elements (ectopic).
endoderm into pouches (Piotrowski and Nusslein-Volhard,
2000). In animals reduced for Fgf8, we observed a range of CB
cartilage phenotypes that suggests defects in the segmentation
of NCC into distinct branchial arches. These phenotypes
include reductions in the number of CB cartilages (Fig. 3B)
and fusions of cartilages of adjacent segments (Fig. 3C);
incompletely formed CB cartilages were also observed (Fig.
3C). In addition, as reported for fgf8– animals (Roehl and
Nusslein-Volhard, 2001), more posterior pharyngeal pouches
were reduced and disorganized in fgf8-MO; fli1-GFP animals
(Fig. 2C,E,G). We investigated whether the CB cartilage defects
seen in fgf8-MO animals might be secondary to posterior pouch
formation defects that result in reduced branchial NCC
segmentation.
In order to understand the cellular basis of NCC
segmentation, we made time-lapse recordings of pharyngeal
arch development in wild-type (see Movie 1 in supplementary
material) and fgf8-MO (see Movie 2 in supplementary material)
animals. Time-lapse recordings were made of wild-type animals
expressing both the pan-nuclear H2A.F/Z:GFP and the NCCexpressing fli1-GFP (12-38 hpf; Movie 1). In wild-type animals,
NCC migrated in three streams to ventrolateral regions, where
they contributed to the formation of the pharyngeal arches.
Starting at 12 hpf (5-somites), H2A.F/Z:GFP-labeled NCC
were seen migrating in two streams (mandibular and hyoid)
anterior to, and one stream (branchial) posterior to, the
developing otic vesicle. By around 16 hpf, the NCC finished
their migration and began to express fli1-GFP as they condensed
to form the arch masses. Shortly after the initiation of fli1-GFP
expression in NCC, the first branching of the branchial mass
occurred as the third pouch was formed (Fig. 3D). Over the next
20 hours, the fourth, fifth and sixth pouches formed in an
anterior-posterior (AP) wave of development, and by 38 hpf the
branchial mass had been subdivided into the five segments from
which the CB cartilages would arise (Fig. 3E-G).
By contrast, in the fgf8-MO; fli1-GFP example shown (see
Movie 2 in supplementary material), one fewer branchial
segment formed. We found that in this animal the reduced
number of segments was due to the failure of the fourth pouch
to develop. While the third pouch (i.e. the first pouch to
subdivide the branchial mass) formed normally (Fig. 3H), the
fourth pouch initiated outgrowth yet failed to fully subdivide
the branchial mass into a new segment (Fig. 3I). By 38 hpf the
fifth and sixth pouches had fully formed, but the fourth pouch
had retracted, and what in wild-type animals would have been
the second and third posterior branchial segments had fused
together, resulting in one less branchial segment (Fig. 3J,K).
Consistent with this animal forming one less branchial
segment, we found that one less CB cartilage developed (data
not shown). These results suggest that at least some, and
possibly all, of the CB cartilage defects seen in fgf8– animals
are the result of a failure of posterior endodermal pouches to
form properly and segment branchial NCC into discrete
arches.
Fgf signaling is required during early somite stages
for first pouch development
In order to determine when Fgfs act to control pouch
development, we inhibited Fgf signaling at different times of
development using the Fgf receptor antagonist SU5402. As
extended (24-hour) treatment of embryos with SU5402
causes widespread death of NCC (David et al., 2002), we
performed shorter treatments of SU5402 in order to dissociate
requirements for Fgf signaling in pharyngeal pouch formation
from those in NCC survival. After addition of SU5402 for 1to 4-hour periods, followed by a washout, embryos were
scored for pouch defects at 34 hpf. In the case of 4-hour
treatments, we examined effectiveness of inhibition of Fgf
signaling by expression of the Ets factor, pea3, in treated
siblings. pea3 is a downstream target of Fgf signaling (Roehl
and Nusslein-Volhard, 2001), and we observed partial-tocomplete inhibition of pea3 expression during the treatment
and gradual recovery after washout (data not shown,
summarized in Fig. 4 legend). In embryos treated with
SU5402 from 10 to 14 hpf, we found that the first pouch was
specifically lost in 39% of embryos (Fig. 4B) and misshapen
in another 26% of embryos (data not shown). In addition,
when animals with first pouch defects were raised to 4 days,
we observed specific losses of hyoid cartilage (Fig. 4E). By
contrast, SU5402 treatments from 6 to 10 hpf and 14 to 18
hpf had lesser effects on pouch development (Fig. 4G). We
found a similar temporal requirement for Fgf signaling
using 1-hour SU5402 treatments (Fig. 4C,H). The highest
penetrance of first pouch shape defects was seen when 1-hour
SU5402 treatments began between 9 and 13 hpf. Interestingly,
later (>20 hpf) treatments with SU5402 produced variable
5708 Development 131 (22)
defects in the development of more posterior pouches but
not the first pouch (Fig. 4F), consistent with more posterior
pouches forming later in an AP temporal wave of
development. In summary, we find that Fgf signaling has a
peak requirement in first pouch development from 10 hpf
(tailbud) to 14 hpf (10-somites).
Neural and mesodermal requirement for Fgfs in
pharyngeal pouch formation
As Fgf8 and Fgf3 are expressed in neural and mesodermal
tissues at early somite stages, and then in the pharyngeal
endoderm at later stages, we investigated which Fgf sources
are required for pouch formation. Using transplantation
techniques (see Materials and methods), we introduced wildtype tissues at pre-shield stages (<6 hpf) into fgf8–; fgf3-MO;
fli1-GFP embryos and then assayed for rescue of first pouch
Research article
development based on GFP-labeled arch structure. First, we
found that wild-type endoderm was not able to rescue first
pouch structure (Fig. 5B-B′′) compared with control
contralateral sides that did not receive transplants (Fig. 5AA′′). Wild-type mesoderm (Fig. 5C-C′′) or neural tissue (Fig.
5D-D′′) alone was able to only partially rescue pouch and arch
structure in less than half of fgf8–; fgf3-MO; fli1-GFP hosts
(Fig. 5F). By contrast, transplantation of wild-type neural and
mesodermal tissues together completely rescued pouch
structure in 3/6 embryos, and partial rescue was seen in another
2/6 embryos (Fig. 5E-E′′). As neural tube transplants alone
were sufficient to rescue the ear and cerebellar defects of fgf8–;
fgf3-MO embryos (Fig. 5D′′′), yet failed to completely rescue
pouch structure (Fig. 5D′), we conclude that the requirements
for both neural and mesodermal tissues is not simply a function
of restoring neural structure in fgf8–; fgf3-MO embryos. Thus,
Fig. 3. Fgf8 is required for segmentation of the branchial arches. Bilateral flat-mount dissections of Alcian-stained pharyngeal cartilages at 4
days. As shown for wild type (A), M and PQ are mandibular (1) cartilages, CH and HS are hyoid (2) cartilages, and CB1-5 cartilages are
formed from the five most posterior branchial arches (3-7). Note the teeth (*) on the CB5 cartilages. fgf8– animals have variable CB cartilage
defects, which include reduced CB number (only 4 CBs per side in B), incompletely formed CB cartilages (arrowhead in C), and fusions
between adjacent cartilages (arrow in C). In a representative fgf8–; fli1-GFP clutch (n=172) there was an average of 3.9 CB cartilages per side;
6% of sides had fusions of adjacent cartilages and 2% had incomplete cartilages. (D-K) Time-lapse recordings of wild-type fli1-GFP;
H2A.F/Z:GFP (D-G, and see Movie 1 in supplementary material) and fgf8-MO; fli1-GFP (H-K, and see Movie 2 in supplementary material)
animals show the cellular basis of branchial arch segmentation. In wild-type animals, branchial arches form as pouches separate the branchial
NCC mass into segments in an A–P wave of development. At the beginning of Movie 1 (5-somites, 12 hpf), H2A.F/Z:GFP labels the nuclei of
NCC that are migrating ventrolaterally in two streams anterior to, and one stream posterior to, the developing ear. After fli1-GFP initiates in
NCC of the pharyngeal arches, selected projections from Movies 1 and 2 show the subdivision of each successive branchial arch (arch 3 at 20
hpf: D,H; arch 4 at 28 hpf: E,I; arch 5 at 34 hpf: F,J; and arches 6 and 7 at 38 hpf: G,K). Pouches are labeled p1-p6, and white arrowheads in
D-G indicate the developing vasculature that also expresses fli1-GFP. The white arrows in panels I-K and Movie 2 (in supplementary material)
refer to arches 4 and 5, which fail to separate completely in this fgf8-MO animal. Similar cell behaviors were seen in three time-lapse
recordings of wild-type animals, and variable defects were observed in four time-lapse recordings of fgf8-MO; fli1-GFP animals. Anterior is to
the left in all panels. A-C are ventral views, and dorsal is up and slightly to the right in D-K. M, Meckel’s cartilage; PQ, palatoquadrate; CH,
ceratohyal; HS, hyosymplectic; CB, ceratobranchial. Scale bar: 50 µm.
Fgfs pattern pouches and cartilages 5709
Fgf8 and Fgf3 are required additively in both the neural tube
and mesoderm, but not the endoderm, to promote
morphogenesis of the pharyngeal endoderm into pouches.
Fgf8 and Fgf3 are required for the subsequent
development, and not the generation, of pharyngeal
endoderm and NCC
In order to test whether the lack of pouches is due to a general
reduction in pharyngeal endoderm, we examined early markers
of pharyngeal endoderm in fgf8–; fgf3-MO animals. In wildtype embryos, the first pouch began to form around 16 hpf (see
below). At 18 hpf, nkx2.7 and axial normally were expressed
in lateral pharyngeal endoderm, in particular the first pouch
and regions where the second and more posterior pouches
would form (Fig. 6A,E). In fgf3-MO animals, nkx2.7 and axial
expression was similar to that seen in wild-type animals (Fig.
6C,G), whereas in fgf8– animals nkx2.7 and axial were present
but first pouch staining was variably absent (Fig. 6B,F).
Strikingly, in fgf8–; fgf3-MO animals, these markers revealed
that a significant amount of pharyngeal endoderm was present,
yet there was no clear evidence of pouches (Fig. 6D,H). In
addition, as assayed by axial staining, we saw no differences
in the amount of pharyngeal endoderm at an earlier stage (10
hpf) between fgf8–; fgf3-MO and wild-type animals (data not
Fig. 4. Fgf signaling is required during early somite stages for first pouch and hyoid cartilage development. (A-C) Confocal micrographs show
Zn8-labeled pharyngeal pouches (red) and GFP-labeled NCC (green) in fli1-GFP animals at 34 hpf. Cranial sensory ganglia (dots) also stain
with Zn8. In the wild-type animal (A), an arrowhead marks the first pouch. (B) The first pouch is variably absent (arrowhead) in fli1-GFP
animals upon treatment with the Fgf receptor antagonist SU5402 from 10-14 hpf. The absence of the first pouch is selective, as more posterior
pouches form normally. (C) Treatment with SU5402 for 1-hour periods starting from 9-13 hpf (a 10.5-11.5 hpf treatment is shown) produce
subtler shape changes of the first pouch (arrowhead). (D,E) Flat-mount preparations of Alcian-stained pharyngeal cartilages at 4 days. In wildtype (D), mandibular M and PQ, hyoid CH and HS, and branchial CB1-5 cartilages are labeled. In those animals in which 10-14 hpf SU5402
treatment caused losses of the first pouch early, the HS cartilage was selectively absent later (E). Although M, PQ and CH cartilages are
reduced in size, posterior CB cartilages are relatively unaffected. (F) Whereas later treatments with SU5402 (a 20-21 hpf treatment is shown) do
not affect first pouch development (arrowhead), they do occasionally disrupt the formation of more posterior pouches (arrow shows an
unsegmented branchial NCC mass). (G) Quantitation of first pouch defects after 4-hour treatments with SU5402. The percentages of animals
with first pouch losses, in black, and misshapen first pouches, in gray, are plotted. n6-10 hpf=49, n10-14 hpf=99, n14-18 hpf=21. First pouch loss after
10-14 hpf treatment is statistically significant using Tukey HSD test. (H) The percentage of fli1-GFP animals having first pouch defects
(primarily shape changes) plotted against the start time of 1 hour treatments with SU5402. n5.5 hpf=17, n7 hpf=14, n8 hpf=24, n9 hpf=14, n10.5 hpf=21,
n11 hpf=18, n12 hpf=26, n13 hpf=26, n14 hpf=22, n16 hpf=26, n20 hpf=24, n24 hpf=31. The period of strongest effect is from 9 hpf (90% epiboly) to 13 hpf
(8-somites). In order to assess the efficiency of inhibition of Fgf signaling, and the recovery after washout, we fixed sibling controls and
examined pea3 expression, a downstream effector of Fgf signaling, at 0 and 4 hours after washout. We know that SU5402 is at least partially
being washed out as omission of the washout step leads to severe necrosis of animals. For 4-hour incubation experiments, the levels of pea3 in
individual animals, relative to those in similarly staged untreated controls, were as follows: 6-10 hpf, 6/8 reduced at 10 hpf, 11/13 reduced and
2/13 absent at 14 hpf; 10-14 hpf, 5/13 reduced and 8/13 absent at 14 hpf, 6/13 reduced and 7/13 absent at 18 hpf; 14-18 hpf, 5/12 reduced and
7/12 absent at 18 hpf. As pea3 levels were similarly reduced at 18 hpf in 10-14 hpf and 14-18 hpf treatments, yet only 10-14 hpf treatments
cause first pouch defects, we conclude that Fgf signaling is required from 10-14 hpf for first pouch development. However, these experiments
do not exclude additional requirements for Fgf signaling at later times. Anterior is to the left and dorsal is up. M, Meckel’s; PQ, palatoquadrate;
CH, ceratohyal; HS, hyosymplectic; SU, SU5402. Scale bar: 50 µm.
5710 Development 131 (22)
shown). These results suggest that Fgf8 and Fgf3 act to
promote the segmentation of pharyngeal endoderm into
pouches and not endoderm generation.
Similarly, in order to understand the losses of the NCCderived cartilages in fgf8–; fgf3-MO animals, we examined
the development of NCC in doubly reduced animals. In 18
Research article
hpf wild-type embryos, dlx2 expression marked three
postmigratory NCC masses: the mandibular, hyoid and
branchial primordia (Fig. 6I). In fgf3-MO and fgf8– embryos,
the three dlx2-positive masses resembled those in wild-type
animals (Fig. 6J,K). By contrast, in fgf8–; fgf3-MO embryos,
only two dlx2-positive masses were present (Fig. 6L). Based
Fig. 5. Fgfs are required in neural and mesodermal tissues
for first pouch formation. Labeled wild-type tissues (red:
A′′-E′′) were transplanted into fgf8–; fgf3-MO; fli1-GFP
animals from 4-6 hpf, and GFP-expressing NCC (green: A′E′) were examined at 34 hpf for rescue of pharyngeal arch
structure, a proxy for pouch structure. A′-E′ and A′′-E′′ are
confocal projections and are merged in A-E. A′′′-E′′′ are
individual confocal sections from A-E and include the
Nomarski channel. (A-A′′′) As transplantations generally contribute donor tissue unilaterally, we used contralateral non-recipient sides of fgf8–;
fgf3-MO; fli1-GFP animals as negative controls for rescue (the red staining in A,A′′ represents a comparatively small amount of donor tissue
that has crossed the midline). In control sides, only mandibular (1) and a few unidentified (?) NCC are evident (A′), and the ear is missing
(A′′′). (B-B′′′) Wild-type endoderm (e) fails to rescue pharyngeal arch structure (B′) and the ear (B′′′). As seen in B′′, wild-type endoderm does
not segment into pouches in fgf8–; fgf3-MO; fli1-GFP hosts. Wild-type mesoderm (m, C-C′′′) or wild-type neural tissue (n, D-D′′′) only
partially rescues pharyngeal arch structure in a fraction of animals. In the non-rescued mesodermal example shown (C′), pharyngeal (1 + ?)
NCC remain unsegmented, revealing a lack of pouches. The neural example shown (D′) represents what we scored as partial rescue of arch
structure. There is an increase in the amount and organization of NCC, but they are not segmented into ordered pharyngeal arches as in wildtype animals. The lack of rescue of arch structure by wild-type neural tissue is striking, as other structures such as the MHB blood vessel
(asterisk in D′), the neural flexure (white arrowhead in D′′), and the ear (black arrowhead in D′′′) are rescued by neural tissue. By contrast,
wild-type mesoderm did not rescue the ear (C′′′). (E-E′′′) Both wild-type mesoderm and neural tissue are required together to completely rescue
pharyngeal arch structure, and hence pouches, in fgf8–; fgf3-MO; fli1-GFP animals. In this example, a morphologically normal first pouch (p1,
arrow) and mandibular (1) and hyoid (2) arches are clearly seen (E′). Some of the more posterior pouches (E′, note the segmentation of the
branchial (3+) NCC mass) and the ear (black arrowhead in E′′′) are rescued as well. The identification of mesoderm in the transplants was
based on the lack of colocalization with the neural crest marker fli1-GFP in confocal sections, and in this example by the characteristic
morphology of the mesodermal cores of the pharyngeal arches (Kimmel et al., 2001) (F) Quantitation of pharyngeal arch rescue by wild-type
tissues is plotted as percentage of host sides with complete (black) or partial (gray) rescue of arch structure. nendoderm=34, nmesoderm=11,
nneural=30, nneural+mesoderm=6. Complete rescue by neural and mesodermal tissue (neur. + meso.) and partial rescue by mesoderm or neural tissue
were statistically significant using Tukey HSD test. In addition, no rescue was seen by wild-type neural crest, a tissue that does not express
either Fgf8 or Fgf3 (data not shown). Anterior is to the left and dorsal is up. Scale bar: 50 µm.
Fgfs pattern pouches and cartilages 5711
Fig. 6. Pharyngeal endoderm and cranial NCC defects in animals lacking Fgf8 and Fgf3. nkx2.7 (A-D) and axial (E-H) label pharyngeal
endoderm during early pouch morphogenesis stages (18 hpf). nkx2.7 and axial are in blue, and, in E-H, krox-20 in red labels R3 and R5. In wildtype animals (A,E), the first pouch (p1: arrows) has formed anterior to R3, and a more posterior endodermal mass that will give rise to the
remaining pouches (black lines) is situated adjacent to R4-R6 territory. The first pouch is variably lost in fgf8– animals (asterisk in B, question
mark in F). Whereas pharyngeal endoderm develops normally in fgf3-MO animals (C,G), in fgf8–; fgf3-MO animals (D,H) pharyngeal endoderm
is present as a single anterior mass (black line) and no pouches are evident. (I-P) dlx2, in blue, labels cranial NCC; in red (I-L), pax2a labels the
MHB and krox-20 labels R3 and R5. (I) In 18 hpf wild-type animals, mandibular (1), hyoid (2), and branchial (3) NCC streams give rise to seven
pharyngeal arches. (M) At 33 hpf, the third branchial stream has generated arches 3-5 and arches 6 and 7 have yet to separate. In fgf8– (J,N) and
fgf3-MO (K,O) animals, the migration and coalescence of NCC to form the pharyngeal arches is largely normal. In fgf8–; fgf3-MO animals, the
mandibular (1) stream is disorganized and hyoid and branchial streams are fused together (2/3) at 18 hpf (L). By 33 hpf (P), nearly all hyoid and
branchial NCC are absent, and mandibular (1) NCC are present but reduced. Anterior is to the left in all panels. A-D are dorsal views, and E-P
are lateral views. R3 and R5, rhombomeres 3 and 5; MHB, midbrain-hindbrain boundary; p1, first pouch. Scale bar: 50 µm.
on their positions with respect to the R3 domain of the
hindbrain, we interpreted these two masses as a disorganized
mandibular mass and a single fused mass incorporating hyoid
and branchial NCC. As hyoid NCC are generated adjacent to
R4 and branchial NCC develop adjacent to R6-R7 domains
(Schilling and Kimmel, 1994; Trainor and Krumlauf, 2001),
the NCC fusions are probably due to the absence of
intervening R5-R6 territory in fgf8–; fgf3-MO embryos
(Maves et al., 2002; Walshe et al., 2002). We observed similar
dlx2 phenotypes at 12 hpf (data not shown). By 33 hpf, dlx2
labeled the mandibular and hyoid arches and four branchial
segments in wild-type animals (Fig. 6M). In fgf3-MO and
fgf8– animals, dlx2 expression was only mildly reduced
compared with wild-type controls (Fig. 6N,O). However, dlx2
expression in fgf8–; fgf3-MO animals revealed that by 33 hpf
most NCC were absent except for a reduced mandibular
population (Fig. 6P). We observed similar NCC losses in fgf8–;
fgf3-MO animals based on the NCC expression of the fli1-GFP
transgene (Fig. 1D). Moreover, the selective disappearance of
hyoid and branchial NCC between 18 hpf and 33 hpf was
consistent with the later specific losses of the hyoid and
branchial cartilages in fgf8–; fgf3-MO animals. In conclusion,
we found requirements for Fgf8 and Fgf3 in both the early
organization (12-18 hpf) and the later survival (33 hpf) of
NCC.
Pharyngeal pouches form by the lateral migration of
endodermal cells
Understanding the role of Fgfs in pouch formation requires a
detailed knowledge of the cell behaviors underlying pouch
development in wild-type animals. Surprisingly, little is known
about the mechanism of pouch formation in any species. In
order to investigate the morphogenesis of pouch endoderm
directly, we made time-lapse recordings of Alexa568-labeled
developing endoderm in H2A.F/Z:GFP; fli1-GFP zebrafish
(10-30 hpf: see Movies 3, 4 in supplementary material). As
labeled endoderm was generated by a combination of TAR*
induction and transplantation techniques (see Materials and
methods for details), a large fraction, but not all, of the
endodermal cells can be seen in the recordings. H2A.F/Z:GFP
labels the nucleus of every cell (Pauls et al., 2001) and helps
to identify landmarks, whereas fli1-GFP labels postmigratory
NCC. At 10 hpf of wild-type development, Alexa568-labeled
pharyngeal endodermal cells were scattered along the surface
of the yolk (Fig. 7A,A′), a distribution that closely resembled
endodermal axial expression at this time (Reiter et al., 2001).
Over the next 6 hours, endodermal cells underwent a medial
migration and became increasingly packed together near the
midline (Fig. 7B,B′ show a 14 hpf intermediate stage). Shortly
after medial migration, endodermal cells that would give rise
to the first pouch began to extend thin cytoplasmic processes
5712 Development 131 (22)
and migrate back out laterally (18 hpf: Fig. 7C,C′ and more
clearly in Movie 4). At this time, the first postmigratory NCC
began to turn on fli1-GFP. During the next few hours,
endodermal cells continued to migrate laterally in a directed
fashion, and by 22 hpf the first pouch was nearly fully formed
(Fig. 7D,D′). In addition, clusters of endodermal cells situated
periodically along the AP axis migrated laterally to form
progressively more posterior pouches in an AP wave of
Research article
development. By 30 hpf in this recording, the positions of the
first three pouches were clearly seen relative to the fli1-GFPlabeled NCC of the arches (Fig. 7E,E′). In three wild-type
recordings, the lateral migration of endodermal cells was
observed to underlie the formation of all labeled pouches. We
conclude that the directed lateral migration of periodically
spaced endodermal cells is the mechanism that generates the
pharyngeal pouches.
Fig. 7. Pharyngeal pouches form by an Fgf-dependent lateral migration of endodermal cells. (A-E) Representative still images from a timelapse confocal recording of pharyngeal pouch and arch development in wild-type animals (see Movies 3, 4 in supplementary material).
Pharyngeal endoderm has been labeled in red (A′-E′; merged with GFP in A-E) by transplanting TAR* endoderm into a fli1-GFP;
H2A.F/Z:GFP host at 4 hpf; this technique leads to mosaic labeling of endoderm in the host animal. In green, H2A.F/Z:GFP allows the nuclei
of every cell to be seen, and fli1-GFP marks NCC of the pharyngeal arches. In wild-type development, endodermal cells are spread out in a
monolayer over the surface of the yolk at 10 hpf (A,A′). Concomitant with medial ectodermal movements to form the neural keel, endodermal
cells migrate medially and begin to aggregate (14 hpf: B,B′). Shortly after medial migration, individual endodermal cells destined to become the
first pouch (arrow in C′) then migrate back out laterally (C: 18 hpf). As seen in Movie 4, pouch endodermal cells extend cytoplasmic processes
laterally during migration. At the same time, cranial NCC begin to condense and express fli1-GFP. By 22 hpf, the first two pouches (D′: p1,p2)
have formed and interdigitate three NCC-containing pharyngeal arches (D: 1-3). Although in this example most second pouch cells are not
labeled in red, their development can still be observed based on transient expression of fli1-GFP (asterisks in C,D). Also, the characteristic
flexure of the neural keel near the MHB is visible by H2A.F/Z:GFP (arrowhead in D). At 30 hpf, three pouches (E′: p1-p3) and four arches (E:
1-4) are well developed. (F-J) Representative still images from a time-lapse recording of pharyngeal development in an animal reduced for Fgf8
and Fgf3 (see Movies 5, 6 in supplementary material). Similar cell behaviors were seen in two separate recordings. Labeled endoderm (red)
was generated by transplantation of wild-type TAR* endoderm into fgf8–; fgf3-MO; H2A.F/Z:GFP animals (see text for discussion of
experimental rationale, including how wild-type and mutant endoderm probably behave similarly in a mutant host). In fgf8–; fgf3-MO animals,
the generation (F,F′) and medial migration (G,G′) of pharyngeal endoderm is normal. However, by 18 hpf, the lateral migration of endodermal
cells is delayed (H,H′). Also, the migration of endodermal cells is disorganized, with cytoplasmic processes not being oriented laterally as in
wild-type animals (arrows in Movie 6 in supplementary material). By 22 hpf lateral endodermal cells form an extended anterior mass (white
line in I′). A confirmation of the fgf8–; fgf3-MO phenotype is the lack of a neural flexure (arrowhead in I), increased cell death at the MHB, and
the lack of an ear (data not shown). By 30 hpf, pharyngeal endoderm has not segmented into discrete pouches and remains a single anterior
mass (white line in J′). Although animals did not carry the fli1-GFP transgene, reduced mandibular (1) and possibly hyoid (2?) arches are
visible as condensations of H2A.F/Z:GFP-expressing cells. The views are dorsolateral with anterior to the left. Scale bar: 50 µm.
Fgfs pattern pouches and cartilages 5713
Fgfs are required for the segmentation and directed
migration of endodermal cells that form pouches
In order to understand the cellular basis for the lack of pouches
in animals reduced for Fgf8 and Fgf3, we made time-lapse
recordings of pharyngeal endoderm development in fgf8–; fgf3MO; H2A.F/Z:GFP animals (10-30 hpf: see Movies 5, 6 in
supplementary material). For technical reasons, we visualized
pharyngeal endoderm by transplanting labeled, TAR*-induced
endoderm from wild-type donors into fgf8–; fgf3-MO;
H2A.F/Z:GFP hosts (see Materials and methods). However, as
we knew that wild-type endoderm failed to make pouches in
an fgf8–; fgf3-MO background, we inferred that the endoderm
defects we describe here would be the same as in non-mosaic
fgf8–; fgf3-MO animals. At 10 hpf, we saw a similar
distribution of pharyngeal endodermal cells over the surface of
the yolk as in wild-type animals (Fig. 7F,F′), consistent with
our earlier finding that fgf8–; fgf3-MO animals had no defect
in 10 hpf endodermal axial expression. In addition, the medial
migration and subsequent compaction of endodermal cells near
the midline was largely normal in fgf8–; fgf3-MO animals (Fig.
7G-H′). However, by 18 hpf we saw defects in the lateral
migration of endodermal cells (Fig. 7H,H′). Although
endodermal cells extended cytoplasmic processes in fgf8–;
fgf3-MO animals, these processes were not always directed
laterally and often retracted (see Movie 6 in supplementary
material). By 22 hpf, endodermal cells had failed to migrate
laterally and discrete pouches were not seen (Fig. 7I,I′).
Moreover, putative pouch endodermal cells did not align into
regularly spaced arrays and by 30 hpf had formed a single,
unsegmented clump of cells laterally (Fig. 7J,J′). We conclude
that Fgf8 and Fgf3 are not required for the initial generation or
medial migration of pharyngeal endodermal cells. Instead, Fgfs
have later functions in the directed lateral migration and regular
spacing of pharyngeal endodermal cells along the AP axis,
processes critical for the formation of discrete pouches.
Discussion
Pharyngeal pouches form by an Fgf-dependent
lateral migration of endodermal cells
We have demonstrated an essential role for Fgf signaling in the
formation of pharyngeal pouches. Whereas fgf8– animals had
variable defects in pouch formation, animals reduced for both
Fgf8 and Fgf3 lacked all pharyngeal pouches. In addition,
transient inhibition of Fgf signaling with the Fgf receptor
antagonist SU5402 caused similar defects in pouch formation.
This essential function of Fgfs in pouch formation is probably
conserved among vertebrates, as mice hypomorphic for Fgf8
or Fgf receptor 1 (FgfR1) have similar pouch defects to those
in fgf8– zebrafish (Abu-Issa et al., 2002; Trokovic et al., 2003).
Previous to this study, little was known about the cellular
behaviors underlying pouch formation. Pharyngeal pouches
arise as lateral branches of the foregut endoderm. Branching
morphogenesis is a common theme in organogenesis, and
various cellular mechanisms, such as clefting and cell
migration, have been implicated in branch generation (Chuang
and McMahon, 2003; Ghabrial et al., 2003; Sakai et al., 2003).
By directly imaging pouch formation in developing zebrafish
embryos, we showed that cell migration is the mechanism that
drives the lateral branching of the pharyngeal endoderm into
pouches. After coalescence of pharyngeal endoderm near the
ventral midline, subsets of endodermal cells migrated laterally
at periodic intervals along the AP axis to form pouches. As
cells could be seen extending cytoplasmic processes laterally
during migration, we propose that chemotactic or substrate
cues in the local environment guide pouch endodermal cells to
lateral positions.
Several lines of evidence indicate that the lack of pouches
in fgf8–; fgf3-MO animals is probably due to a defect in the
later morphogenesis of pouch endoderm. Based on the
expression of endodermal markers such as axial and nkx2.7,
pharyngeal endoderm was present in fgf8–; fgf3-MO animals,
although we do not know if mediolateral patterning of the
endoderm was completely normal. Whereas axial and nkx2.7
expression suggest that pouch formation was defective at early
stages in fgf8–; fgf3-MO animals, a lack of pouch formation
was clearly seen later using the Zn8 antibody or transplant
techniques to label endoderm. A role for Fgfs in the
morphogenesis of pouch endoderm was most evident in timelapse recordings of endodermal development in fgf8–; fgf3-MO
embryos. Whereas the generation, medial migration and
coalescence of endodermal cells were largely normal, we saw
defects in both the later lateral migration and AP positioning
of pharyngeal endodermal cells in fgf8–; fgf3-MO embryos.
The migration of endodermal cells was delayed, and the thin
cytoplasmic processes characteristic of migrating cells were
disorganized in fgf8–; fgf3-MO embryos. Thus, in the absence
of Fgfs, putative pouch endodermal cells had the ability to
migrate but could not orient themselves along the mediolateral
axis. In addition, whereas in wild-type embryos pouch
endodermal cells migrated laterally at periodic AP positions,
in fgf8–; fgf3-MO embryos endodermal cells remained a
continuous mass occupying the anterior pharyngeal region. We
propose that Fgf signaling may regulate both the migration and
AP positioning of pouch endodermal cells, and future
experiments are needed to elucidate the extent to which these
processes are interrelated.
How do neural and mesodermal Fgfs pattern
pharyngeal pouches?
Our transplantation experiments demonstrated that Fgf8 and
Fgf3 are required additively in the neural keel and head
mesoderm to rescue first pouch formation in fgf8–; fgf3-MO
embryos. Consistent with this, inhibition of Fgf signaling from
tailbud (10 hpf) to 10-somites (14 hpf), stages at which Fgf8
and Fgf3 are expressed in the neural keel and lateral mesoderm,
blocked first pouch formation. An attractive model is that
signals from the neural keel help to pattern both the pharyngeal
endoderm and premigratory NCC (Fig. 8A). Such a strategy
would link the two sources of pharyngeal segmentation,
segmentation of NCC into distinct streams and segmentation
of the endoderm into pouches, to the earlier segmentation of
the hindbrain. Intriguingly, mandibular NCC originate from
ectomesenchyme adjacent to the MHB and are Hox-negative,
and the first pharyngeal pouch develops in the vicinity of
MHB-R2 and is also Hox-negative (Hunt et al., 1991; Miller
et al., 2004). By contrast, the second pouch and hyoid NCC
develop adjacent to R4 and are both Hox-positive. Whereas
development of the first two pouches connects to segmental
expression of Fgf in the hindbrain, it is less clear how
development of the more posterior pouches would be
regulated. All pouches were lost in animals lacking both Fgf8
5714 Development 131 (22)
Research article
Fig. 8. Fgf8 and Fgf3 as positional determinants of
pharyngeal segmentation. Model of pharyngeal
segmentation in wild type (A,B) and animals lacking Fgf8
and Fgf3 (C,D). This model is based on fgf8 expression
(Reifers et al., 2000) and fgf3 expression (Maves et al.,
2002) at 13 hpf (8-somites). A and C are lateral views with
anterior to the left and dorsal up, and B and D are crosssectional views at the level of R2. (A,B) In wild type, Fgf8
protein, dark blue, is produced in neural MHB-R2 and R4
domains and in the lateral mesoderm. Fgf3 protein, light
blue, is co-produced with Fgf8 in the MHB and R4 (striped
domains represent overlap). Mandibular (1) NCC (Hox
negative: light green) are generated adjacent to MHB-R2
territory, whereas hyoid (2) and branchial (3) NCC (Hox
positive: dark green) have their origins at R4 and R6-R7 axial levels, respectively. Likewise, the first (p1) endodermal pouch (Hox negative:
red) develops ventrolateral to R2, and the second (p2) and more posterior (p3+) pouches (Hox positive: wine) form ventrolateral to R4 and R6R7, respectively. The ear (black circle with two dots) develops adjacent to R5. (B) A cross-sectional view shows that during lateral migration
pouch endodermal cells are in close proximity to Fgf-expressing ventral neural keel and lateral mesoderm. Pharyngeal pouches would form
where Fgf expression in the hindbrain coincides with Fgf8 expression in the underlying lateral mesoderm. (C,D) In fgf8–; fgf3-MO animals,
NCC and pharyngeal endoderm are generated but subsequent morphogenesis is defective. Pharyngeal endoderm remains unsegmented and
hyoid and branchial NCC streams fuse. The structure of the hindbrain is also defective in animals lacking Fgf8 and Fgf3. MHB, R1, R5 and R6
regions fail to develop, and R2 and R3 are reduced in size (Brand et al., 1996; Walshe et al., 2002; Maves et al., 2002; Reifers et al., 1998). As
Fgfs are required in both neural and mesodermal tissues to promote the formation of pouches, Fgf signaling may link early neural and
mesodermal patterning to segmentation of the pharyngeal endoderm. In a direct model, Fgfs from the lateral mesoderm and ventral hindbrain
act as chemoattractants to promote the lateral migration of pouch endodermal cells (B). In animals lacking Fgf8 and Fgf3, pouch endodermal
cells would fail to get the appropriate cues to migrate laterally (D). Alternatively, in an indirect model, Fgfs function to regulate the structure of,
and gene expression in, the hindbrain and lateral mesoderm. In the absence of Fgf signaling, guidance cues for pouch endodermal migration
would be reduced or absent. LM, lateral mesoderm; MHB, midbrain–hindbrain boundary; R, rhombomere.
and Fgf3, and transient inhibition of Fgf signaling, at times
later than those used to inhibit first pouch formation, disrupted
the formation of more posterior pouches. However, pouches
three through six develop at stages when Fgfs are no longer
expressed in the hindbrain and head mesoderm. Further
analysis will be required to determine how similar the
functions of Fgfs in posterior pouch formation are to those
described here for first pouch formation.
Although we showed that Fgfs are essential for pouch
morphogenesis, our results do not distinguish between direct
and indirect functions of Fgfs in pouch outgrowth. For
example, Fgfs from the lateral mesoderm may act directly as
chemoattractants for the lateral migration of pharyngeal
endodermal cells. In Drosophila, the branching of the trachea
also requires Fgf signaling (Klambt et al., 1992; Sutherland
et al., 1996), and tracheal cells have been shown to migrate
toward ectopic sources of Fgf (Sato and Kornberg, 2002).
Alternatively, Fgfs may influence pouch formation indirectly
by regulating patterning of the hindbrain and lateral mesoderm.
By 16 hpf, pharyngeal endodermal cells in close proximity
ventrally to the neural keel and medially to lateral mesoderm
begin to migrate to form the pouches (Fig. 8B). However, we
could block first pouch formation by inhibiting Fgf signaling
from 10-14 hpf, although we cannot rule out that there is a
delay between addition of the drug and effective inhibition of
Fgf signaling. In one model, the role of Fgfs would be to
establish segmental signals in the hindbrain that control the
later lateral migration of endodermal cells at periodic positions
along the AP axis. Consistent with this, animals reduced for
Fgf8 and Fgf3 have hindbrain defects that include losses of
MHB and segments R1, R5 and R6, and reductions in size of
additional rhombomeres (Walshe et al., 2002; Maves et al.,
2002). If pouch endodermal cells are responding to segmental
cues in the hindbrain for their migration, the reduced size and
segmentation of the hindbrain may explain why endodermal
cells did not migrate at discrete AP positions and ultimately
formed compressed clumps in fgf8–; fgf3-MO animals (Fig.
8C,D). In addition, as fgf8– but not fgf3-MO zebrafish have
defects in MHB structure (Reifers et al., 1998), a lack of early
Fgf8-dependent MHB signals might explain why fgf8– but not
fgf3-MO embryos had variable first pouch defects. Similarly,
Fgf8 has been shown to control both the gene expression
profile and morphogenesis of the lateral head mesoderm
(Reifers et al., 2000). Thus, Fgfs might promote pouch
formation indirectly by controlling the positioning and
expression of pouch guidance factors in the hindbrain and
lateral mesoderm. Future experiments that address where Fgf
signaling is required, for example by manipulating Fgf receptor
function in the endoderm and other tissues, should help to
clarify how directly Fgfs act to control pouch formation.
Pharyngeal pouches pattern cartilages of the hyoid
and branchial arches
In fgf8–; fgf3-MO animals, no pouches formed and little or no
cartilage was made from the Hox-expressing NCC of the hyoid
and branchial arches. Similarly, transient inhibition of Fgf
signaling during early somite stages led to correlated losses of
both the first pouch and dorsal hyoid cartilage. However, in
fgf8–; fgf3-MO animals, mandibular cartilages, which are
derived from NCC that do not express Hox genes, were less
affected. By contrast, cas mutant zebrafish lack all endoderm
and are missing cartilages derived from all pharyngeal arches
(David et al., 2002), implying that Fgf-independent
endodermal signals pattern cartilages of the mandibular arch.
As pharyngeal endoderm was present but not segmented into
pouches in fgf8–; fgf3-MO animals, we conclude that the
Fgfs pattern pouches and cartilages 5715
outpocketing of the pharyngeal endoderm to form pouches is
a critical event that allows endoderm to induce cartilage in
Hox-positive NCC.
By analyzing individual sides of fgf8– animals, we found a
correlation between early changes in pouch structure and later
rearrangements of the cartilage pattern. In our studies of
integrinα5 mutant zebrafish, we found that the first pouch
promotes the local compaction and survival of NCC that give
rise to specific regions of dorsal hyoid cartilage (Crump et al.,
2004). In fgf8 mutant sides in which the first pouch was shifted
in position or an ectopic pouch formed in presumptive hyoid
NCC territory, we observed similar positional shifts of
dorsal hyoid cartilage or ectopic cartilage elements. These
correlations are consistent with the abnormal first pouch
promoting hyoid cartilage development in abnormal locations.
In other cases a deformed first pouch invaded NCC territory
that, based on our previous fate mapping (Crump et al., 2004),
normally forms dorsal hyoid cartilage, and this deformed
pouch was correlated with a subsequent loss of dorsal hyoid
cartilage. In addition, defects in the formation of more
posterior pouches were correlated with losses and fusions of
the ceratobranchial cartilages. Thus, whereas early pouch
defects are largely predictive of later cartilage alterations, the
precise interpretation of the resultant cartilage defects in fgf8–
animals is complicated by the fact that Fgf8 probably has
multiple functions in pharyngeal cartilage development.
Our finding that pharyngeal pouches were essential sources
of patterning information for the cartilages of the Hoxexpressing hyoid and branchial arches is consistent with work
in chicken showing that different types of foregut endoderm
interact with Hox-expressing versus non-Hox-expressing NCC
to specify cartilage pattern. In these studies, involving the
transplantation and ablation of endoderm domains at stages
prior to pouch morphogenesis, anterior endoderm can respecify
cartilage pattern when transplanted adjacent to Hox-negative,
but not Hox-positive, NCC (Couly et al., 2002). However, more
posterior endoderm can respecify cartilages derived from Hoxpositive NCC (Ruhin et al., 2003). Based on our analysis of
Fgf function in zebrafish, we predict that the posterior
endoderm domains that induce cartilage from Hox-positive
NCC in chicken will include endodermal regions that form
pharyngeal pouches during later embryogenesis.
Lastly, what are the pouch-derived factors that promote
cartilage development? Recent evidence suggests that Fgfs
themselves are expressed later in the pouches and promote the
survival of skeletogenic NCC. Studies in zebrafish have shown
that Fgf3 is required in the pharyngeal endoderm for the
survival of hyoid and branchial NCC (David et al., 2002;
Nissen et al., 2003). As Fgf8 is also expressed, albeit less
strongly, in the pouches, it has been proposed that Fgf8 may
act redundantly with Fgf3 as an endoderm-derived NCC
survival factor (Walshe and Mason, 2003a). Interestingly, we
did observe graded reductions of dorsal hyoid cartilage in some
fgf8– animals that had normal first pouches, consistent with
Fgf8 also having a role in the later survival of NCC. Moreover,
the lack of hyoid and branchial cartilages in fgf8–; fgf3-MO
animals is most consistent with a survival defect of
postmigratory Hox-positive NCC. Based on dlx2 expression,
hyoid and branchial NCC are present early but disappear later
in fgf8–; fgf3-MO animals. However, as pouches do not form
in fgf8–; fgf3-MO animals, the NCC survival defects are
probably due in part to there being no pouches to secrete
survival factors such as Fgf8 and Fgf3. As has been observed
in tooth and lung development (Chuang and McMahon, 2003;
Jernvall and Thesleff, 2000), it is becoming apparent that Fgfs
also have multiple, temporally distinct functions during
pharyngeal ontogeny. We propose that, in addition to a later
function as pouch-derived survival factors, an essential early
function of Fgfs in endodermal pouch morphogenesis may help
explain the diversity of craniofacial phenotypes seen in fgf8animals.
We thank John Dowd and the UO Fish Facility for abundant help
with raising fish; Jose Campos-Ortega for providing the
H2A.F/Z:GFP line before publication; Craig T. Miller, Le Trinh, Nick
Osborne and Tom Schilling for helpful discussions, especially about
endoderm; and Johann Eberhart for comments on the manuscript.
J.G.C. is an O’Donnell Fellow of the Life Sciences Research
Foundation. L.M. was supported by a fellowship from the Damon
Runyon-Walter Winchell Cancer Research Fund. Research is funded
by NIH grants DE13834 and HD22486.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/131/22/5703/DC1
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